The basic hallmarks of bronchial asthma, one of the most common chronic diseases occurring in the world, are chronic inflammation, remodelling of the bronchial wall and its hyperresponsiveness to environmental stimuli. It was found out that the fibroblast to myofibroblast transition (FMT), a key phenomenon in subepithelial fibrosis of the bronchial wall, was crucial for the development of asthma. Our previous studies showed that HBFs derived from asthmatic patients cultured in vitro display some inherent features which facilitate their TGF-b-induced FMT. Although usefulness of standard ‘2D’ cultures is invaluable, they have many limitations. As HBFs interact with extracellular matrix proteins in the connective tissue, which can affect the FMT potential, we have decided to expand our ‘2D’ model to in vitro cell cultures in 3D using collagen gels. Our results showed that 1.5 mg/ml concentration of collagen is suitable for HBFs growth, motility, and phenotypic shifts. Moreover, we demonstrated that in the TGF-β1-activated HBF populations derived from asthmatics, the expression of fibrosis-related genes (ACTA2, TAGLN, SERPINE1, COL1A1, FN1 and CCN2) was significantly increased in comparison to the non-asthmatic ones. We also confirmed that it is related to the TGF-β/Smad2/3 profibrotic pathway intensification. In summary, the results of our study undoubtedly demonstrate that HBFs from asthmatics have unique intrinsic features which predispose them, regardless the culture conditions, to the increased FMT under the influence of TGF-β1.